Our long-term objective is to understand signaling pathways that protect the heart from[unreadable] ischemia/reperfusion (I/R) injury. Our short-term objective, and the topic of this proposal, is to examine the[unreadable] mechanism of cardioprotection by the cytosolic small heat shock protein (sHSP), alphaB-crystallin (alphaBC). alphaBC[unreadable] (22 kDa) is expressed predominantly in cell types with high oxidative phosphorylation demands, e.g. cardiac[unreadable] myocytes. In cardiac myocytes, alphaBC is phosphorylated upon stimulation of stress MAP kinases (e.g. p38),[unreadable] which, if chronically activated, also drive increased alphaBC expression. Increasing the level of wt alphaBC, or[unreadable] expressing pseudophosphorylated alphaBC, protects against I/R injury. Our preliminary data indicate that[unreadable] mitochondria serve as an alphaBC-binding target during I/R. Accordingly, our hypothesis is that phosphorylated[unreadable] alphaBC protects cardiac myocytes from I/R injury and a portion of this protection is mediated by the conditional[unreadable] association of phospho-alphaBC with mitochondrial outer membrane (mOM) proteins, such as the MPTP. The[unreadable] Specific Aims that address this hypothesis are to:[unreadable] 1) examine the kinetics with which I or I/R increase m-alphaBC, and to assess the phosphorylation status of[unreadable] m-alphaBC, using a combination of immunoblotting, confocal and electron microscopy,[unreadable] 2) assess the effects of alphaBC deletion, or overexpression of wild type or mutant forms of alphaBC on MPTP[unreadable] activation, apoptosis, autophagy and myocardial function in response to I/R, and[unreadable] 3) identify mitochondrial alphaBC binding partners and elucidate l/R-dependent changes in the mitochondrial[unreadable] subproteome.[unreadable] Significance and Innovation: The proposed studies are the first to examine the interaction of alphaBC with[unreadable] mitochondria in any tissue type, and the first to study how m-alphaBC preserves mitochondrial function during[unreadable] stress. These studies employ a comprehensive series of experiments using state-of-the-art cellular,[unreadable] molecular genetic and proteomics technologies to discover new information required to understand cellular[unreadable] mechanisms that preserve mitochondrial function during I/R stress.